Abstract
Metformin is a well-tolerated antidiabetic drug and has recently been
repurposed for numerous diseases, including pain. However, a higher
dose of metformin is required for effective analgesia, which can
potentiate its dose-dependent gastrointestinal side effects. Curcumin
is a natural polyphenol and has beneficial therapeutic effects on pain.
Curcumin has been used as an analgesic adjuvant with several analgesic
drugs, allowing synergistic antinociceptive effects. Nevertheless,
whether curcumin can exert synergistic analgesia with metformin is
still unknown. In the present study, the nature of curcumin-metformin
anti-inflammatory interaction was evaluated in in vitro using
lipopolysaccharide-induced RAW 264.7 macrophage and BV-2 microglia
cells. In both macrophage and microglia, curcumin effectively
potentiates the anti-inflammatory effects of metformin, indicating
potential synergistic effects in both peripheral and central pathways
of pain. The nature of the interaction between curcumin and metformin
was further recapitulated using a mouse model of formalin-induced pain.
Coadministration of curcumin and metformin at a 1:1 fixed ratio of
their ED[50] doses significantly reduced the dose required to produce a
50% effect compared to the theoretically required dose in phase II of
the formalin test with a combination index value of 0.24. Besides, the
synergistic interaction does not appear to involve severe CNS side
effects indicated by no motor alterations, no alterations in short-term
and long-term locomotive behaviors, and the general well-being of mice.
Our findings suggest that curcumin exerts synergistic anti-inflammation
with metformin with no potential CNS adverse effects.
Subject terms: Drug discovery, Pain
Introduction
Pain is defined as “an unpleasant sensory and emotional experience
associated with, or resembling that associated with, actual or
potential tissue damage”^[36]1. Though pain serves a protective
function, the excessive triggering of nociceptors by inflammatory
mediators leads to a devastating condition identified as inflammatory
pain^[37]2. Pathophysiology of inflammatory pain is characterized by an
excessive peripheral and central immune response, indicated by
increased release of inflammatory mediators, including cytokines,
chemokines, proteases, and growth factors^[38]3. The activation of
peripheral and central immune cells contributes to enhanced pain
transmission via peripheral and central sensitization,
respectively^[39]3,[40]4. Several treatment options are currently
available to treat inflammatory pain, including non-steroidal
anti-inflammatory drugs, steroids, and opioids. Yet, the side effects
of current treatments, including gastrointestinal irritation,
cardiovascular toxicity, nephrotoxicity, and dependence (opioids),
continued to be an issue^[41]5. Given these issues, searching for
alternative treatments for pain management is imperative.
Metformin is a well-tolerated antidiabetic drug (Fig. [42]1A), and
recently several pre-clinical and clinical studies have shown the
potential analgesic effects of metformin^[43]6–[44]10. Given that this
drug is a disease-modifying agent in type II diabetes mellitus, it is
possible that it has similar properties for reducing pain and its
comorbidities^[45]11. Metformin has been shown to alleviate pain-like
behaviors by activating the AMP-activated protein kinase (AMPK) and
opioidergic pathways^[46]6,[47]12. In rodent models of pain, metformin
was found to inhibit peripheral and central inflammation, major
contributing factors of pain progression, along with reflexive and
non-reflexive pain behaviors^[48]13,[49]14. However, a clinical study
conducted on diabetic patients indicated the necessity of using a
higher dose of metformin in humans to be a potential analgesic^[50]15.
The use of a higher dose of metformin may increase the chance of
experiencing side effects associated with metformin which include most
commonly gastrointestinal disturbances: diarrhea and nausea, abdominal
discomfort and indigestion, and hypoglycemia^[51]16. The undesirable
gastrointestinal side effects of metformin were observed in ~ 25% of
patients, where ~ 5% were unable to tolerate it^[52]17. The side effect
of metformin also has been associated with lactic acidosis^[53]18.
Hence, it is essential to lower the analgesic dose requirement of
metformin, which can be achieved by applying several approaches,
including the drug combination and nanoparticle approaches.
Figure 1.
Figure 1
[54]Open in a new tab
Chemical structures of metformin hydrochloride (A) and curcumin (B).
Curcumin
([1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione)
is a naturally occurring polyphenolic compound (Fig. [55]1B), first
isolated from the herbaceous perennial plant, Curcuma longa (turmeric).
Curcumin has been used as a traditional herbal remedy for centuries
throughout Asia due to its pleiotropic activities, including
anti-inflammatory, antioxidant, and anticancer^[56]19. It is a
well‐tolerated natural product causing no or minimal toxicity in short-
and long-term use^[57]20. Consequently, it was declared “generally
recognized as safe” by the US Food and Drug Administration
(FDA)^[58]21. Moreover, the therapeutic effectiveness of curcumin in
nociceptive, inflammatory, and neuropathic pain has been reported in
numerous animal models and humans^[59]22. Considering its analgesic
mechanisms of action, it effectively attenuates pain by modulating the
neurotransmitters related to pain, suppressing the immune response, or
blocking the transient receptor potential vanilloid type I (TRPV1)
receptors, and modulating purinergic and chemokine
receptors^[60]23–[61]27. However, curcumin has low stability and poor
water solubility and is quickly metabolized in the gastrointestinal
tract and liver, despite its apparent benefits^[62]27.
In the last few years, research has shown that herb-drug combinations
in pain management produce higher efficacy and lesser adverse effects
than a single drug administration^[63]28. This could be due to their
ability to target several sites of the pain pathway, which minimizes
the effective doses of both compounds in the combination. Dual
treatment with curcumin and metformin has been reported in diabetic
mellitus^[64]29, diabetes-induced comorbidities^[65]30,
nephrotoxicity^[66]31, hepatocellular carcinoma, pancreatic cancer
cells, and breast cancer^[67]32, with the results suggesting
synergistic effects^[68]31. Moreover, metformin and curcumin have
different mechanisms of action in pain modulation, which indicates the
potential for exerting greater analgesia when administered together.
Hence, this study aims to evaluate the nature of the pharmacological
interaction between curcumin and metformin in both the peripheral and
central levels of pain transmission to identify their potential use in
analgesia.
Results and discussion
Curcumin, in combination with metformin, synergistically inhibited the NO
production of LPS-stimulated RAW 264.7 macrophage and BV-2 microglial cells
Macrophage and microglia, immune cells located in the peripheral and
central nervous systems (PNS and CNS), respectively, play a major role
in the pathogenesis of inflammatory pain via neuroimmune crosstalk.
Immune cells in the PNS and CNS produce mediators (proinflammatory
cytokines and chemokines) that modulate pain sensitivity. Nociceptor
neurons, in turn, release neuropeptides and neurotransmitters that
regulate the immune cell responses. Moreover, microglia also respond to
proinflammatory signals generated from non-neuronal cells, including
immune cells. Therefore, the crosstalk between nociceptor neurons and
immune cells and between immune cells in the PNS and CNS is a primary
factor affecting both acute and chronic inflammation^[69]33–[70]35.
Accordingly, in vitro screening of compounds in macrophage or
microglial cells has been used to identify the potential analgesic
compounds. Hence, in this study, curcumin and metformin were first
evaluated in vitro, in lipopolysaccharide (LPS)-induced RAW 264.7
macrophage and BV-2 microglial cells to determine the nature of the
interaction between compounds in peripheral and central pathways of
pain.
Initially, the maximum non-cytotoxic concentrations of each compound
alone were determined, which were 5 µM and 10 µM for curcumin in RAW
264.7 and BV-2 cell lines, respectively, and 1 mM for metformin in both
cell lines. Concentrations for combination treatment were determined
based on their safety concentrations: curcumin and metformin ratios of
1:200 and 1:100 for RAW 264.7 and BV-2 cells, respectively. As shown in
Fig. [71]2, none of the treatments at selected concentrations caused
significant cytotoxicity on LPS-stimulated RAW 264.7 and BV-2
microglial cells: the cell viability was greater than 94%.
Figure 2.
[72]Figure 2
[73]Open in a new tab
Cytotoxicity profile of LPS-stimulated RAW 264.7 macrophage (A) and
BV-2 microglial (B) cells with or without treatment of curcumin (CUR),
metformin (MET), or their combination. RAW 264.7 macrophages were
treated with 0.625–5 µM of CUR, 0.125–1 mM of MET, or their combination
at a 1:200 CUR: MET ratio for 12 h, whereas BV-2 cells were treated
with 1.25–10 µM of CUR, 0.125–1 mM of MET or their combination at
a 1:100 ratio of CUR: MET for 24 h. The cell viability was determined
by MTT assay and expressed as a percentage of the control. Experimental
data are presented as mean ± SD, n = 3 independent experiments.
Then the anti-inflammatory effect of curcumin and metformin in
LPS-stimulated RAW 264.7 macrophage and BV-2 microglial cells was
determined using the compounds alone and in combination. Treatment with
LPS induces the release of nitric oxide, one of the main inflammatory
mediators released at the inflammatory sites, into the culture medium.
Secretion of nitric oxide (NO) is mediated via inducible nitric oxide
synthase (iNOS), in which a high level of nitric oxide is identified as
a marker of severe inflammation^[74]36. Therefore, the suppression of
nitric oxide release is considered a plausible approach for the
attenuation of the inflammatory process. The experiment was designed
based on the Chou–Talalay method for synergy determination of drug
combinations using the constant ratio drug model^[75]37. The nitrite
concentration in the culture supernatant was determined as an indicator
of nitric oxide^[76]38.
Curcumin, metformin, and their combination inhibited LPS-induced NO
production in a concentration-dependent manner (Fig. [77]3A,B). When
curcumin concentrations at 1.25–5 µM were combined with metformin
0.25–1 mM, a synergistic response was achieved in RAW 264.7 macrophage
cells. However, in BV-2 cells, only additive or antagonistic effects
were observed at lower concentrations of combination treatment except
for the highest concentration of combination (10 µM CUR and 1 mM MET),
which showed strong synergism (Fig. [78]3C,D). When the LPS-stimulated
cells were treated with the highest concentration of curcumin alone (5
and 10 µM for RAW 264.7 and BV-2 cells, respectively), NO production
decreased only by 41.2 ± 3.4% and 52.7 ± 3.4% in RAW 264.7 and BV-2
cells, respectively. A lower level of inhibition in NO production was
observed when 1 mM of metformin alone was applied to cells: by
13.0 ± 0.8% and 26.4 ± 5.3% in RAW 264.7 and BV-2 cells, respectively.
However, once the combination therapy was put in, NO release reduced
remarkably: by 64.4 ± 3.9% and 89.9 ± 2.2% in RAW 264.7 and BV-2 cells,
respectively.
Figure 3.
[79]Figure 3
[80]Open in a new tab
Anti-inflammatory effects of curcumin and metformin alone and in
combination on peripheral and central immune cells. (A,B) Dose–response
curves of NO inhibitory effect of curcumin and metformin alone or in
combination on LPS-induced RAW 264.7 macrophage (A) and BV-2 microglial
(B) cells. (C,D) Fraction affected (Fa)-combination index (CI) plot for
the combined effect of curcumin and metformin on LPS-induced NO
production in RAW 264.7 (C) and BV-2 (D) cells. Generally, CI < 1,
CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic
interaction, respectively. (E,F) Normalized isobologram for the
anti-inflammatory effect of curcumin and metformin on RAW 264.7
macrophage (E) and BV-2 microglial (F) cells. Experimental data are
presented as mean ± SD, n = 3 independent experiments.
The interaction between curcumin and metformin was then determined by
calculating the combination index value according to the Chou and
Talalay method, where the CI values of 1, > 1, and < 1 referred to
additive, antagonistic, and synergistic interactions,
respectively^[81]37. The CI values of the curcumin-metformin
combination in RAW 264.7 and BV-2 cells were 0.28 ± 0.02 and
0.08 ± 0.01, respectively, which indicate a very strong synergism
between curcumin and metformin in both the cell lines. Moreover, the
IC[50] values of the curcumin-metformin combination in both RAW cells
(3.47 ± 0.35 µM CUR + 0.67 ± 0.07 mM of MET) and BV-2 cells (3.5 µM of
CUR + 0.35 mM of MET) were lower than the IC[50] values obtained for
individual treatment with metformin (10.31 ± 3.48 mM for RAW.264.7 and
2.76 mM for BV-2 cells) and curcumin (13.07 ± 2.24 µM for RAW 264.7 and
8.86 µM for BV-2 cells). To further demonstrate the synergistic
anti-inflammatory effect between curcumin and metformin, an isobologram
was constructed. The experimentally derived IC[50] values of curcumin
and metformin in both cell lines were located below the additivity line
of the isobologram, indicating a synergistic interaction between the
two compounds (Fig. [82]3E,F).
The combination of curcumin and metformin decreased expression levels of
proinflammatory cytokines in LPS-stimulated RAW 264.7 macrophage and BV-2
microglial cells
The overexpression of proinflammatory cytokines is one of the major
factors governing peripheral and central pain sensitization. Hence,
reducing the production of proinflammatory cytokines by both peripheral
immune cells (macrophages) and central immune cells (microglia and
astrocytes) is an effective approach for improving pain
conditions^[83]36,[84]39. Thus, we examined the effect of curcumin and
metformin in combination on the release of LPS-induced proinflammatory
cytokines: IL-6 and TNF-α. When RAW 264.7 macrophage cells were treated
with the highest nontoxic concentrations of curcumin (5 µM) and
metformin (1 mM) alone, curcumin produced 26.2 ± 1.5% inhibition of
IL-6 and 25.7 ± 1.1% inhibition of TNF-α, while metformin alone
produced 11.2 ± 3.1% inhibition of IL-6 and 20.4 ± 0.2% inhibition of
TNF-α. However, treatment with curcumin and metformin in combination
(5 µM and 1 mM, respectively) exerted significantly higher inhibition
of cytokine production compared to the individual treatment:
49.7 ± 0.5% inhibition of IL-6 and 52.4 ± 1.1% inhibition of TNF-α
(Fig. [85]4A,B). In addition, the combination of curcumin and metformin
at lower concentrations (2.5 µM and 0.5 mM, respectively) inhibited
IL-6 expression in 34.6 ± 1.0% and TNF-α expression in 46.5 ± 3.8%,
which are comparable with the additive inhibition of 5 µM curcumin and
1 mM metformin (Supplementary Table [86]1).
Figure 4.
[87]Figure 4
[88]Open in a new tab
Effects of curcumin, metformin alone, and their combination on
proinflammatory cytokine production in peripheral and central immune
cells. (A,B) The effect of 5 µM curcumin, 1 mM metformin, and their
combination on IL-6 (A) and TNF-α (B) expression in LPS-stimulated RAW
264.7 macrophage cells. (C,D) The effect of 10 µM curcumin and 1 mM
metformin and their combination on IL-6 (C) and TNF-α (D) expression in
LPS-stimulated BV-2 microglial cells. Data are expressed as means ± SD
calculated from three independent experiments. *p < 0.05 treatments
compared to LPS only, ^$p < 0.05 compared to the curcumin alone and
^#p < 0.05 compared to the metformin alone.
A similar pattern of inhibition was observed in LPS-stimulated BV-2
microglial cells. The treatment of curcumin (10 µM) and metformin
(1 mM) alone leads to a moderate decrease in cytokine expression:
curcumin caused 53.1 ± 3.0% and 30.4 ± 6.7% inhibition of IL-6 and
TNF-α, respectively, while metformin caused 21.0 ± 3.4% and 29.3 ± 1.7%
inhibition of IL-6 and TNF-α, respectively. However, the combination
treatment of curcumin and metformin (10 µM and 1 mM, respectively)
produced an 87.8 ± 3.8% and 64.4 ± 9.2% inhibition in IL-6 and TNF-α
expression, respectively, which were significantly higher than the
inhibitory effect obtained with individual treatments (Fig. [89]4C,D).
The treatment of cells with a half lower concentration of curcumin and
metformin (5 µM Cur + 0.5 mM Met) exerted 70.8 ± 10.8% inhibition of
IL-6 expression and 29.5 ± 6.6% inhibition of TNF-α expression
(Supplementary Table [90]2).
Curcumin, metformin, and their combination alleviate formalin-induced
inflammatory pain-like behaviors in mice
Following the substantial findings in the in vitro models, we further
expanded the experiments to pre-clinical models using a mouse model of
formalin-induced inflammatory pain. This model is commonly used to
represent pain-like behaviors induced by noxious stimuli. Intraplantar
administration of formalin causes a characteristic biphasic response.
Phase I response is an acute pain behavior mainly caused by the
immediate and extensive excitation of afferent c-fibers, and the latter
phase II response results from inflammatory processes in the peripheral
tissues that lead to peripheral sensitization^[91]40. Despite the
short-term responses, phase II is also characterized by a sustained
inflammatory mediator release due to the activation of spinal
microglia, which leads to sensitization of projection neurons, termed
central sensitization^[92]41–[93]43.
Mice were pre-treated orally with different doses of curcumin or
metformin (3–300 mg/kg) alone or combined at a fixed-dose ratio (1:1)
of their respective ED[50] doses. One-hour post-treatment, 10 µL of 5%
formalin solution was administered subcutaneously to the plantar
surface of the left hind paw, followed by behavioral observation for
40 min (Fig. [94]5A). The formalin-induced pain-like behavior is
indicated as hind paw licking duration (Fig. [95]5A) and frequency
(Supplementary Fig. [96]1). The administration of formalin produced
specific pain-like behavior characterized by licking of the injected
paw, observed in a distinguish biphasic response: early phase I
(0–5 min) followed by an idle period (5–10 min) and thereafter, a late
phase from 10 to 40 min (Fig. [97]5B–D). The hind paw licking behavior
peaked at 0–5 min and 20–30 min post-formalin injection, which then
gradually declined in all groups. As shown in Fig. [98]5A,B, in the
early phase, both curcumin and metformin monotherapy did not show
strong antinociception where they only produced significant
antinociception at higher doses (100 and 300 mg/kg). However, curcumin
and metformin in monotherapy and combination therapy dose-dependently
inhibited the formalin-induced hind paw licking in late phase II
(Fig. [99]5E–G). Hence, the type of interaction between two compounds
was then determined by considering their effect on phase II pain-like
behaviors.
Figure 5.
[100]Figure 5
[101]Open in a new tab
Orally administered curcumin, metformin, and their combination
alleviate the formalin-induced inflammatory pain in mice. (A) Schematic
presentation of the experimental design. (B–D) Time course of
formalin-induced hind paw licking behavior in mice with oral curcumin
(B), metformin (C), and their combination (D). (E–G) The total duration
of hind paw licking during phases I and II of the pain-like behavioral
response with the treatment of curcumin (E), metformin (F), and their
combination (G). Note that curcumin, metformin, and their combination
dose-dependently inhibited phase II response in mice. *p < 0.05,
**p < 0.01 and ***p < 0.001 compared to the vehicle-treated group.
One-way ANOVA followed by Bonferroni’s post hoc test, n = 8 mice per
treatment group.
The formalin-induced first and second-phase responses had unique
characteristics^[102]44. The first phase is due to the direct
activation of nociceptors by formalin (somatic pain). The second phase
is due to inflammatory processes (inflammatory pain), leading to
peripheral and central sensitization. Medicines that act centrally,
such as narcotics, inhibit both stages equally, but those that act
peripherally, such as NSAIDs and steroids, only inhibit the second
phase. Thus, this model enables researchers to distinguish between
somatic and inflammatory pain and central and peripheral analgesic
mechanisms^[103]45. In this study, curcumin and metformin showed
effects in both phases, wherein in phase 1, significant antinociception
was only observed at higher doses (100 and 300 mg/kg). However, more
potent inhibition was observed in phase II licking behaviors. This
indicates the potential contribution of both the PNS and CNS to the
curcumin and metformin antinociception.
Curcumin and metformin produce synergistic antinociception in
formalin-induced mice
Interaction between curcumin and metformin with respect to the
antinociceptive effect exerted by the treatments in the late phase of
the formalin test was analyzed according to the method described by
Tallarida et al.^[104]46. Initially, the log dose–response curves for
the effect of acute oral administration of curcumin, metformin, and
curcumin-metformin combination were constructed (Fig. [105]6A).
Curcumin and metformin showed dose-dependent antinociceptive effects
where the maximum dose evaluated (300 mg/kg) demonstrated the maximal
antinociception: 60% for curcumin and 58% for metformin. The doses
exerting 50% antinociception (ED[50]) were established at
82.8 ± 17.6 mg/kg and 248.9 ± 106.5 mg/kg for curcumin and metformin,
respectively assuming a linear-logarithmic model of the dose–response
curves. As shown in Fig. [106]6A, the dose–response curve obtained for
the curcumin and metformin co-treatment at a 1:1 fixed ratio showed a
dose-dependent effect, and the curve shifted to the left. There was a
remarkable reduction in the ED[50] to 39.6 ± 7.1 mg/kg (ED[50 mix]) and
70% maximum antinociception. The slopes of regression lines for
curcumin and metformin monotherapy were 19.9 ± 2.2 and 20.9 ± 3.4,
respectively. The statistical analysis indicated the parallelism of the
dose–response curves (p > 0.05, student’s t-test). Hence, the type of
interaction between compounds was determined using standard type I
isobololographic analysis.
Figure 6.
[107]Figure 6
[108]Open in a new tab
Isobolographic analysis of the antinociceptive interaction between
curcumin and metformin in phase II of the formalin test. (A) Linear
regression of log dose–response curves of curcumin, metformin, and
curcumin-metformin combination. (B) The isobologram of the
curcumin-metformin interaction at the fixed-dose ratio of 1:1. The
straight line connecting the ED[50]s of curcumin and metformin
represents the additive line. The horizontal and vertical bars
represent the SEM. ED[50] (Cur) and ED[50] (Met) correspond to the
ED[50]s obtained with individual treatment of curcumin and metformin,
respectively. ED[50 add] and ED[50 mix] correspond to the theoretical
and experimentally derived ED[50]s of curcumin and metformin
combination. The ED[50 mix] value lies far below the additive line,
suggesting significant synergism. ***p < 0.001 compared to the ED[50
add,] by student’s t-test.
The isobologram of curcumin-metformin combination indicated synergistic
interaction between curcumin and metformin (Fig. [109]6B) as the
experimentally derived ED[50 mix] located below the theoretical
additive line connecting ED[50] values of curcumin and metformin
monotherapy. Table [110]1 presents the theoretical additive and
experimentally derived ED[50] values for the combination. As indicated
in the table, the experimentally derived ED[50 mix] was substantially
reduced by 76% than the theoretically presumed ED[50 add] (p < 0.001,
student’s t-test). In addition, the combination index of the
curcumin-metformin combination was 0.24. Altogether, these results
suggest the synergistic interaction between curcumin and metformin in
inhibiting phase II pain-like behaviors of formalin-induced mice at the
50% effect level.
Table 1.
50% antinociceptive doses and interaction index of curcumin, metformin,
and curcumin–metformin combination in phase II of mouse formalin test.
Monotherapy ED[50] ± SEM (mg/kg)
Curcumin 82.8 ± 17.6
Metformin 248.9 ± 106.5
Combination therapy (1:1) Ratio ED[50 add] ± SEM (mg/kg) ED[50
mix] ± SEM (mg/kg) CI
CUR + MET 1:3 165.8 ± 62.0 39.6 ± 7.1*** 0.24
Curcumin 41.4 ± 8.8 9.9 ± 1.8
Metformin 124.4 ± 53.2 29.7 ± 5.3
[111]Open in a new tab
The ED[50] values were determined from linear regression analysis of
the log dose–response curves. The theoretical ED[50] (ED[50 add]) was
calculated based on the dose–response curves of the curcumin and
metformin monotherapy. The experimental ED[50] (ED[50 mix]) was
determined by the experimentally derived dose–response curve of the
curcumin–metformin combination treatment. The ED[50]s are presented
with their respective S.E.M. values.
CI combination index.
***p < 0.001 compared to the ED[50 add] (student’s t-test).
Previous studies reported curcumin synergism with other analgesic
drugs, including diclofenac and pregabalin, in animal models of
nociceptive pain^[112]47,[113]48. These previous findings, along with
our study findings, indicate the potential of curcumin to exert
synergistic multimodal analgesia with other drugs, which could be due
to the diverse and complementary mechanisms of action of curcumin. The
analgesic effect of curcumin was explained by its ability to attenuate
neurotransmitters related to pain (substance P), suppress the immune
response, and prostaglandin E2 production by suppressing
cyclooxygenase-2 (COX-2), modulating purinergic and chemokine
receptors, and activate the opioid system^[114]49. The pharmacodynamic
interaction between curcumin and metformin is plausible as metformin
shows a distinct mechanism of analgesia to curcumin: activation of
AMPK, opioidergic mechanisms, and suppressing peripheral and central
inflammation^[115]6,[116]13,[117]14,[118]50. Consequently, metformin
showed synergistic analgesia with several other analgesics, including
ibuprofen, aspirin, tramadol, and pregabalin^[119]51.
Administration of formalin into rodents’ hind paws is also associated
with excitation of sensory neurons via direct activation of cation
channels TRPA1/TRPV1^[120]52,[121]53. Moreover, effective attenuation
of TRP channels by both metformin and curcumin has been reported.
Curcumin was found to regulate TRPV1 channels in vivo via antagonizing
their activation and inhibiting phosphorylation^[122]25,[123]54. In
addition, metformin was also found to modulate TRPV1 channels in a
murine model of bone cancer via decreased ASIC3 and TRPV1
expression^[124]55. Therefore, inhibition of TRP receptors may also
have a major contribution to the curcumin-metformin synergistic
antinociception, which needs to be confirmed in future studies.
Moreover, the synergistic analgesia obtained between curcumin and
metformin could also be due to the pharmacokinetic interaction. Oral
administration of curcumin was reported to inhibit several
drug-metabolizing enzymes, including cytochrome P450: CYP3A4,
glutathione-S-transferase, and UDP-glucuronosyltransferase. Hence, oral
curcumin acts as a bioenhancer for the drugs metabolize via cytochrome
P450 enzymes, including morphine, acetaminophen, and digoxin^[125]56.
Metformin, on the other hand, has a limited probability of eliciting
pharmacokinetic interactions since it isn’t metabolized or bound to
plasma proteins in substantial amounts^[126]57. Hence, in this study,
pharmacokinetic interactions were not evaluated yet cannot be excluded.
The underlying mechanism of curcumin-metformin combination by network
pharmacology analysis
In the present study, the potential underlying mechanism of the
curcumin-metformin combination was investigated using network
pharmacology. Network pharmacology integrates pharmacology and network
biology. It is an approach to providing a comprehensive overview of the
interactions between compounds, targets, and diseases in a holistic
way^[127]58. As shown in Fig. [128]7, there are 414, 76, and 459
potential targets for curcumin, metformin, and curcumin-metformin
combination, respectively. In addition, 866 targets of rheumatoid
arthritis (RA) associated genes were retrieved from the databases. The
Venn diagram shows that the intersection target genes between
rheumatoid arthritis and curcumin, metformin, and the
curcumin-metformin combination were 74, 11, and 78, respectively
(Fig. [129]7A). The interactions between compounds and inflammatory
pain-related genes were also constructed using the Cytoscape. As shown
in network interaction, some genes were targeted by both curcumin and
metformin (Supplementary Fig. [130]2).
Figure 7.
[131]Figure 7
[132]Open in a new tab
Network pharmacology analysis of underlying mechanisms of
curcumin-metformin combination in inflammatory pain. (A) The
intersection between the test compounds (curcumin, metformin, and
curcumin-metformin combination) and rheumatoid arthritis-related
targets. (B) Protein–protein interaction of 76 intersection genes (B)
and the top 10 hub gene-network interactions (C). Gene ontology (D–F)
and KEGG (G) enrichment pathway analyses. CUR curcumin, MET metformin,
MET-CUR metformin-curcumin combination, GO Gene Ontology, KEGG Kyoto
Encyclopedia of Genes and Genomes.
Intersection targets between curcumin-metformin combination and
inflammatory pain-related diseases (RA) were analyzed for
protein–protein interactions using the STRING database. The results
demonstrate complex interactions between the involved genes. In the
network, there were 78 nodes and 785 edges constructed. Furthermore,
the PPI network was analyzed using CytoHubba (maximal clique centrality
(MCC) analysis to obtain 10 top interactions between genes. The top 10
genes include AKT1, ALB, CASP3, EGFR, JUN, MMP2, MMP9, PTGS2, STAT3,
and TNF (Fig. [133]7C). These targets potentially play a major role in
the effects of the curcumin-metformin combination on inflammatory pain.
The illustrations of biological mechanisms and pathways were obtained
after GO and KEGG enrichment analyses (Fig. [134]7D–G). The top five
biological processes were significantly enriched in the regulation of
inflammatory processes, responses to bacterial molecules,
lipopolysaccharide, oxygen species metabolic process, and oxidative
stress. These biological processes play a major role in inflammatory
pain, indicating the potential ability of the curcumin-metformin
combination to alleviate inflammatory pain (Fig. [135]7D). Furthermore,
the top five cellular components were significantly enriched in the
membrane raft, membrane microdomain, membrane region, vesicle lumen,
and cytoplasmic vesicle lumen (Fig. [136]7E).
For the molecular function, the activities of endopeptidase,
metalloendopeptidase, nuclear receptor, ligand-activated transcription
factor, and serine-type peptidase were substantially enriched
(Fig. [137]7F). To illustrate the potential pathways of
curcumin-metformin in the treatment of inflammatory pain, the potential
targets were analyzed for KEGG pathway enrichment analysis. The top
five pathways include TNF signaling pathway, lipid and atherosclerosis,
fluid shear stress and atherosclerosis, PD-L1 expression and PD-1
checkpoint pathway in cancer, and toxoplasmosis (Fig. [138]7G). Here,
we found that inflammatory pathways, especially the TNF-signaling
pathway, are the main pathways involved in the anti-inflammatory
effects of the curcumin-metformin combination. The TNF-signaling
pathway has been associated with inflammatory pain, especially in
rheumatoid arthritis. The blockage of TNF-alpha resulted in the
improvement of pain-like behaviors^[139]59. Therefore, the ability of
the combination to suppress inflammatory pain could be attributed to
its ability to modulate immune cells and inflammatory pathways.
Synergistic attenuation of pain-like behaviors by curcumin and metformin is
not accompanied by CNS side effects
In light of the antinociceptive synergism between curcumin and
metformin in the formalin test, we evaluated the effects of individual
compounds and their combination on motor coordination and short-term
locomotor activity in naïve male-ICR mice. Measurement of locomotor
activity allows us to determine whether the combination has potential
side effects, either sedative or CNS stimulative^[140]60,[141]61. The
compounds in monotherapy or combination therapy were tested at the
highest doses used in the formalin test (Fig. [142]8A). The effect of
drug combination on forced locomotive behavior was evaluated using the
rotarod test. The mouse’s ability to remain on a rotating rod (18 rpm)
was measured periodically at 30-, 60-, 90-, 120- and 240-min
post-treatments. As shown in Fig. [143]8B, curcumin and metformin alone
or in combination showed no effect on motor coordination, balance, and
muscle relaxation in the animals. Moreover, the effect of compounds on
short-term spontaneous locomotor activity was measured by using an
automated home-cage monitoring system, Laboratory Animal Behavior
Observation, Registration and Analysis System (LABORAS). Compounds
alone or combined were orally administered 1 h before the behavioral
measurements, and the behavioral measures were taken for 30 min
(Fig. [144]8A). Mice treated with either curcumin and metformin alone
or in combination explored throughout the home cage as indicated by
position distribution in Fig. [145]8C. Neither curcumin and metformin
monotherapy nor curcumin-metformin combination caused significant
alterations in short-term spontaneous locomotive behaviors (locomotion,
climbing, and rearing) compared to that of the control group (p > 0.05)
(F[146]ig. [147]8D–I). The vehicle-treated mice engaged in locomotor
behavior mainly during the first 15 min of the experiment. In contrast,
during the latter period of the experiment (15–30 min), time spent in
immobility was increased. This behavioral pattern is most likely due to
the exploratory behaviors followed by adaptation to the new home cage
environment. The same behavioral stereotype was observed with the
treatment of curcumin and metformin alone or in combination, with no
significant difference in behaviors compared to the vehicle-treated
group.
Figure 8.
[148]Figure 8
[149]Open in a new tab
Effects of curcumin and metformin alone or in combination on forced and
spontaneous locomotor activity of naïve mice. (A) Schematic
presentation of the experimental design. (B) Effect of treatments on
rotarod performance (n = 6/group). (C–I) Short-term locomotive
behaviors, measured by LABORAS 1 h post-treatment for 30 min (n = 8
mice/group). (C) Position distribution of mouse inside the home cage.
(D) Time spent on locomotion. (E) Average speed in locomotion. (F)
Distance traveled in each 5 min. (G–I) Time spent on climbing, rearing,
and immobility, respectively. Data are presented as mean ± S.E.M.
Short-term locomotive behavioral data were analyzed over 5-min
intervals during 30 min test session.
Curcumin, metformin, and their combination showed no significant effect on
the general behavior and well-being of mice
The general behavior and well-being data in rodents can be translated
to CNS side effects in humans^[150]62. The different behaviors of
rodents, including locomotor activity and rearing, immobility, and food
intake/body weight, are used to define dizziness, somnolence, and
nausea in humans, respectively^[151]62. Hence, curcumin and metformin
alone and their combination were evaluated on LABORAS for 24 h to
assess their effects on the general behavior of mice. Mice were
administered with either curcumin and metformin alone or in combination
at the highest doses-tested, placed individually in the LABORAS home
cages, and behavioral measures were recorded for 24 h. Mice treated
with the vehicle spent more time on mobile behaviors during the
nighttime compared to the daytime (Fig. [152]9). This behavioral
pattern is expected as the mice are nocturnal animals. The time spent
on and frequency of mobile behaviors, including locomotion, climbing,
and rearing, gradually declined for 12 h. (18.00–6.00), slightly
increased at the beginning of the daytime (6.00–8.00), and nearly no
mobility was observed thereafter, which was then started to increase by
the end of the test period (16.00–18.00). In line with that, immobility
was lower during the nighttime and higher during the daytime
(Fig. [153]9A–H). Distance traveled by vehicle-treated mice inside the
cage also showed the same pattern with mobile behaviors (Fig. [154]9I).
Moreover, the vehicle-treated mice showed a maximum average speed of
77 mm/s, which remained static during the nighttime, gradually declined
during the daytime, and then reached the same constant level by the end
of the experiment. The same behavioral pattern was observed with the
treatment of compounds in monotherapy or combination (Fig. [155]9).
Neither individual nor combination treatment significantly affected on
long-term spontaneous locomotive behavior in mice observed during the
day and nighttime.
Figure 9.
[156]Figure 9
[157]Open in a new tab
Effects of curcumin and metformin alone or in combination on the
general behavior of naïve mice. Behavioral measures were recorded for
24 h post-treatment using LABORAS. (A,B) Time spent on locomotion (A)
and frequency of its occurrence (B). (C,D) Time spent on climbing (C)
and frequency of its occurrence (D). (E,F) Time spend on rearing
behavior (E) and frequency of its occurrence (F). (G,H) Time spent on
immobility (G) and frequency of its occurrence (H). (I) Distance
traveled by mouse in the cage. (J) Average speed in locomotion. Data
are presented as mean ± S.E.M (n = 10 mice/group). Behavioral data were
analyzed over 2 h intervals during 24 h test sessions.
The body weight and food and water intake are also considered measures
of general well-being in rodents. Hence, we evaluated the effects of
compounds in monotherapy or combination on those parameters during the
24 h LABORAS test period. As indicated in Fig. [158]10A, mice in the
vehicle group showed an average weight loss of 4.4%, wherein treatment
groups showed no significant difference in the weight loss compared to
the vehicle-treated group. The weight loss observed in all the
treatment groups, including the vehicle-treated group, could be
attributed to the isolation of animals during the LABORAS experiment.
Generally, mice are identified as social creatures. During this
experiment, mice must be individually placed in the LABORAS cage where
they were originally housed as 5 mice/cage. Hence, the isolation of
mice from their peers could stress out the animals leading to weight
loss. Moreover, the vehicle-treated group showed average food and water
intake of 3.6 g and 6.9 mL, respectively, for 24 h. The values obtained
with the treatment of curcumin and metformin alone or in combination
showed no significant difference from the vehicle-treated group
(Fig. [159]10B,C). These results indicate that curcumin and metformin
in monotherapy or combination had no effect on the general well-being
of mice.
Figure 10.
[160]Figure 10
[161]Open in a new tab
Percentage weight loss, food intake, and water intake of mice after
24 h treatment with curcumin and metformin alone or in combination. The
weight of the mice and food and water volume supplied to each cage were
measured pre-, and post-test (24 h). The percentage weight loss (A) and
food intake (B), and water intake (C) were calculated and presented as
mean ± S.E.M (n = 10 mice/group).
Most of the available analgesics are reported to possess CNS side
effects. For example, NSAIDs are associated with drowsiness, cognitive
dysfunction, and psychotic disorders^[162]63,[163]64; opioids are
associated with psychomotor and cognitive impairment, drowsiness, and
sleep disturbances^[164]65,[165]66. In addition, gabapentanoids are
reported to cause sedation and cognitive effects^[166]67, and CNS side
effects of cannabinoids are depression, sedation, euphoria, and
psychosis^[167]68. Thus, analgesics with a higher safety margin are a
constant challenge in drug development. Interestingly, our study
reports no potential CNS side effects of curcumin and metformin in
combination, suggesting its possible use in clinical trials. However,
further studies in humans are warranted to ensure the efficacy and
safety of the curcumin-metformin combination in the management of pain.
Conclusion
In summary, this study suggests for the first time that curcumin
combined with metformin exerts synergistic anti-inflammatory effects in
both in vitro and in vivo conditions. Curcumin synergistically
augmented the inhibition of nitric oxide and proinflammatory cytokines
by metformin both in RAW 264.7 macrophage and BV-2 microglial cells.
Besides, the in vivo experiments through formalin-induced mice verified
the synergistic analgesic effects. Moreover, the combined therapy using
curcumin and metformin showed no considerable CNS adverse effects in
naïve mice. Hence, this study supports the possibility of combined use
of curcumin and metformin in the treatment of pain with the least
amount of medication while taking the easiness of administration, cost
of the therapy, and side effect profile of medicines into the account.
Methods
Cell culture
RAW 264.7 macrophage cells (ATCC, Rockville, MD, USA) and BV-2
microglial cells (Accegen Biotechnology, New Jersey, USA) were cultured
in 75 cm^2 flasks in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 1%
penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cultured
flasks were incubated at 37 °C in a 5% CO[2] humidified atmosphere. The
medium was replaced every 2–3 days, and cells were sub-cultured by
trypsin treatment twice a week. RAW 264.7 macrophage (200,000
cells/well) and BV-2 microglial cells (150,000 cells/well) were seeded
in 24-well plates in DMEM supplemented with 10% FBS and incubated
overnight. On the next day, cells were pre-treated for 2 h with
curcumin, metformin, or combination, followed by incubation with
lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA). RAW cells
were incubated with 100 ng/mL LPS for 12 h, and BV-2 cells with 1 µg/mL
LPS for 22 h. Then the culture supernatant was collected to analyze
nitrite level and cytokine production by NO assay and ELISA,
respectively. Curcumin was kindly provided by the Natural Products for
Ageing and Chronic Diseases Research Unit, Faculty of Pharmaceutical
Sciences, Chulalongkorn University, and metformin by the Siam Bheasach
Co. Ltd. (Bangkok, Thailand). All experiments were performed in
triplicate, and results are mentioned as mean ± SD from three
independent experiments.
Cytotoxicity assay
Twenty-four hours after preconditioning of seeded cells, the medium was
replaced with 500 µL of serum-free DMEM media containing different
concentrations of curcumin (1.25–20 µM) and metformin (0.25–10 mM) or
their combination. After 24 h treatments, media were replaced by MTT
(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)
solution (0.5 mg/mL in PBS), and the plates were incubated at dark for
2 h at 37 °C with 5% CO[2] humidified atmosphere. Then the MTT
containing media was removed, and the formed formazan crystals were
dissolved by adding DMSO to each well. Finally, the plates were shaken
for 10 min in the dark, followed by an absorbance measurement at 570 nm
using a microplate reader.
NO assay
The nitrite production in the culture supernatant was determined by the
Griess reaction. Briefly, 100 µL of culture supernatant was transferred
to a 96-well plate, and 50 µL of 1% [w/v] sulfanilamide was added and
incubated for 5 min in the dark. Then, 50 µL of 2.5% [w/v]
N-1-Napthylenediamine dihydrochloride was added and incubated for 5 min
in the dark. The absorbance was recorded at 520 nm. The concentration
of NO was calculated by comparison with a standard curve of sodium
nitrite. The percentage inhibition of NO production in treated cells
was expressed as the percentage of absorbance of LPS-treated cells,
considered 100% NO production.
Median-effect analysis: computerized simulation by CompuSyn
The type of interaction between curcumin and metformin on RAW 264.7 and
BV-2 cells was determined according to the median-effect principle
described by Chou et al.^[168]37 using the CompuSyn software^[169]69.
In Chou and Talalay method, the interaction between compounds in the
mixture is determined based on the median effect equation, which
describes dose–effect relationships:
[MATH: Fa/Fu=C/Cmm, :MATH]
1
where F[a] is the fraction affected by C, F[a] ranges from 0 to 1,
F[a] = 0 and 1 represent 0% and 100% inhibition of NO production
compared to the LPS control, respectively; F[u] is the fraction
unaffected (F[a] + F[u] = 1); C is the concentration of compound; C[m]
is the concentration required to inhibit the NO production by 50%, and
m is the sigmoidicity coefficient of the dose–effect curve. The
combination index (CI) that indicates the interaction between two
compounds is then determined using the following formula:
[MATH: CI=C1/Cx1+C2/Cx2, :MATH]
2
where C[1] and C[2] are the concentrations of compound 1, and compound
2 in combination required to produce x% of effect; [C[x]][1] and
[C[x]][2] are the concentrations of individual compounds required to
produce the same x% effect. The type of interaction between compounds
was determined by the Fa-CI plot. CI values of 1, > 1, and < 1 referred
to additive, antagonistic, and synergistic interactions.
ELISA test
The expression levels of TNF-α and IL-6 in the culture supernatant were
measured using commercial enzyme-linked immunosorbent assay (ELISA)
kits, as indicated by the manufacturer (BioLegend, San Diego, CA, USA).
The absorbance was measured at 450 nm. The concentration of cytokines
in the culture supernatant was determined from their respective
standard curves.
Animals
Male ICR mice at the age of 4–5 weeks were purchased from Nomura Siam
International Co., Ltd., Bangkok, Thailand. Animals were housed (4–5
mice per cage) under controlled temperature (24 ± 2 °C), humidity
(60 ± 10%) and light (12:12 h light/dark cycle), and free access to
food and water at the animal facility, Faculty of Pharmaceutical
Sciences, Chulalongkorn University, Thailand. Animals were allowed to
acclimatize to laboratory conditions for at least one week before the
test procedures. Ethical approval was obtained from the Institutional
Animal Care and Use Committee, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok, Thailand, before the commencement of
the study (Protocol No. 2033006). All the tests were conducted in
compliance with the ARRIVE guidelines (Animal Research: Reporting of In
Vivo Experiments).
Drugs and treatments
In treatment groups, curcumin or metformin was orally administered at
doses of 3, 10, 30, 100, and 300 mg/kg; mice in each control group
received 0.5% carboxymethylcellulose (CMC). To determine the
interaction between curcumin and metformin, mice were then administered
with different doses of curcumin and metformin mixed at a fixed ratio
(1:1) of their respective ED[50] dose for the individual treatment:
1/16, 1/8, 1/4, and 1/2 (metformin ED[50] dose + curcumin ED[50] dose).
All treatments were suspended in 0.5% CMC and given orally at 10 mL/kg.
The number of animals for each experimental group was calculated using
G*Power 3.1.9.6 software at 5% type I error (α = 0.05), 80% statistical
power (1 − β = 0.8), and 0.5 effect size.
Formalin test in mice
Before the nociceptive induction, mice were orally administered
different treatments (8 mice per group) and placed in acrylic chambers
for 1 h for acclimatization to the experimental setup. Behind each
acrylic chamber, two mirrors were placed at a 45° angle to facilitate
the behavioral observations. Then each mouse received 10 µL of 5% (v/v)
formalin in normal saline to the plantar surface of the left hind
paw^[170]70. Immediately after formalin administration, mice were
placed back in the acrylic chambers, and pain-like behaviors were
recorded for 40 min. Then the recorded videos were analyzed using the
Behavioral Observation Research Interactive Software (BORIS)^[171]71,
and the duration of hind paw licking in each 5 min was determined. Hind
paw licking in 0–5 min was considered early phase I, wherein 10–40 min
period was considered later phase II. The percentage of antinociception
in either phase was calculated using the following formula:
[MATH: %Antinociception=100-Dtreatment/Dcontrol×100
, :MATH]
3
where D[treatment] is the duration of hind paw licking for each treated
mouse, and D[control] is the average time of hind paw licking in the
vehicle-treated group.
Analysis of interaction between curcumin and metformin
The interaction between two compounds was determined according to the
method described by Tallarida et al.^[172]72. Briefly, dose–response
curves for individual treatments and combination were constructed using
least-squares linear regression, and the dose resulting in 50% of the
effect (ED[50] ± SEM) was calculated. The theoretical ED[50] for
curcumin-metformin combination was calculated by assuming the
occurrence of additive interaction between compounds by using the
following equation:
[MATH: ED50(add)=fED50(cur)+1-fED50(met), :MATH]
4
where ED[50 (add)] is the theoretical ED[50]; ED[50 (cur)] and ED[50
(met)] are experimental ED[50] of curcumin and metformin, respectively;
and f is the fixed ratio of each compound (0.5). Then the theoretical
and experimental ED[50] (ED[50 (mix)]) values for the combination were
compared using the t-test. The interaction was interpreted as additive
if the ED[50 (add)] and ED[50 (mix)] were not significantly different.
If the ED[50 (mix)] was significantly lower or higher than the ED[50
(add),] the interaction was defined as synergistic or antagonistic,
respectively. Moreover, the type of interaction between two compounds
was also demonstrated by calculating combination index (CI) and
isobologram analysis. The CI was calculated using the following
equation:
[MATH: CI=ED50(mix)/ED50(add). :MATH]
5
The interaction between two compounds was defined as synergistic,
additive, and antagonistic if CI < 1, CI = 1, and CI > 1, respectively.
Moreover, the type of interaction was also demonstrated by an
isobologram as described by Tallarida et al. Briefly, the ED[50] doses
of curcumin and metformin were plotted as axial points in a Cartesian
plot, and then the ED[50 (add)] and ED[50 (mix)]. Then a straight-line
connecting ED[50 (met)] and ED[50 (cur)] was plotted (additive line).
The type of interaction was determined by the location of the ED[50
(mix)] relative to the additive line.
Network pharmacology analysis
The SMILES and SDF formats of curcumin and metformin were obtained from
PubChem ([173]https://pubchem.ncbi.nlm.nih.gov) and further used to
investigate their respective potential targets in several databases,
including Swiss Target Prediction
([174]http://www.swisstargetprediction.ch/), Pharmapper (PharmMapper
(lilab-ecust.cn)^[175]73, and Similarity ensemble approach (SEA Search
Server (bkslab.org)^[176]74. These databases can predict the targets of
curcumin and metformin at the cellular and mechanistic levels.
Furthermore, inflammatory pain-related targets were used to identify
the potential targets in inflammatory pain. Since rheumatoid arthritis
is the most common prevalence of inflammatory pain, the keyword of
“rheumatoid arthritis” was applied for finding related target genes in
several databases, including the DisGeNET database (Fit score ≥ 0.1)
([177]https://www.disgenet.org/), OMIM^® database
([178]https://www.omim.org/), GeneCards (Score ≥ 10)
([179]https://www.genecards.org/). Homo sapiens was selected as the
target species. The target genes obtained from all databases were
further unified and standardized using the UniProt database
([180]https://www.uniprot.org/), and the duplicate targets were then
removed.
The intersection between the target genes of curcumin, metformin, and
curcumin and metformin combination and inflammatory pain-related
targets was assessed using Venny 2.1
([181]https://bioinfogp.cnb.csic.es/tools/venny/). The intersection of
genes of curcumin-metformin combination and genes of target diseases
were further used as a potential target of curcumin-metformin
combination on inflammatory pain. The protein–protein interaction was
performed and constructed using the STRING database and exported into
Cytoscape software (3.9.1) for further analysis. The cytoHubba v.0.1
plugin of Cytoscape was then used to determine the top 10 genes having
a high degree of interactions. To illustrate the potential mechanism of
action and pathways, enrichment analysis was performed using Gene
Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analyses. The KEGG analysis was performed as
previously described^[182]75. GO and KEGG pathway enrichment analyses
were performed and visualized using the Online tool of bioinformatic
data analysis ([183]http://www.bioinformatics.com.cn/).
Rotarod test in mice
Three days before the test, mice were trained to remain on a rotating
rod at a constant speed (18 rpm) for 180 s every day^[184]76. On the
experiment day, mice were tested before drug administration, and mice
capable of remaining on the rod for 180 on three successive trials were
used for the subsequent experiment. Four independent animal groups (6
mice per group) were examined for motor coordination after oral
administration of either vehicle, the highest dose of curcumin,
metformin or curcumin-metformin combination. The latency to fall was
recorded at 30-, 60-, 90-, 120-, and 240-min post-treatment with the
cut-off value of 180 s.
Exploratory behaviors by LABORAS
The effect of test compounds on the short-term locomotive behaviors was
assessed using an automated home-cage monitoring system, LABORAS
(Metris, Hoofddorp, Netherlands). LABORAS system picks up vibrations
generated by the movements of the rodent and converts those to the
behavioral classifications, including climbing, rearing, locomotion,
and immobility^[185]77. The experimental setup was prepared by adding
corn cob bedding to each LABORAS cage (22 cm × 16 cm × 14 cm). Then
mice were orally administered with the vehicle, the highest dose of
curcumin, metformin (300 mg/kg), and their combination (Met 124.4 + Cur
41.4 mg/kg) (8 mice/group). One hour after, mice were placed in the
LABORAS cage, and locomotive behavior was recorded for 30 min. The
effect of each compound on short-term locomotive behavior was
determined separately for each behavioral measure and presented as a
cumulative value of both behavioral duration and frequency.
General behaviors by LABORAS
LABORAS system facilitates the automatic measurement of rodents’
activity in an undisturbed environment for longer durations. Mice were
treated with either vehicle or the highest dose of curcumin, metformin,
or their combination (10 mice/group), and placed on LABORAS cages
supplemented with food and water. Behavioral measurements were started
at 18.00 and recorded for 24 h until 18.00 the next day, both in light
and dark cycles. Behavioral analysis was subsequently divided into 2 h
intervals. After every experiment, the bedding material, food, and
water were removed and replaced after cleaning the cages properly with
70% alcohol.
Statistical analysis
The data analysis was performed using GraphPad Prism 9.1 (GraphPad
Software Inc., La Jolla, CA, USA). All the collected data were
summarized with counts and percentages for categorical variables. The
numerical values were presented as mean ± SEM. The difference between
groups was determined by the One-way analysis of variance (ANOVA)
followed by the post hoc test. Statistical significance was considered
to be achieved when the p-value was < 0.05 (95% confidence level).
Ethics declarations
All the animal protocols were approved by the Institutional Animal Care
and Use Committee (IACUC) of the Faculty of Pharmaceutical Sciences,
Chulalongkorn University (Protocol No. 2033004) and carried out in
accordance with the recommendations of the IACUC. All the tests were
reported in compliance with the ARRIVE guidelines (Animal Research:
Reporting of In Vivo Experiments).
Supplementary Information
[186]Supplementary Information.^ (6.1MB, docx)
Acknowledgements